The invention relates to the application of chromatographic separation methods by the size exclusion principle (size exclusion chromatography; SEC) to continuous chromatography methods, in particular to simulated moving bed (SMB) chromatography.
The separation of substances, in particular macromolecules, by molecular size is a widespread chromatographic separation principle. Particularly in the purification of peptides and proteins, methods such as size exclusion chromatography (SEC), frequently also referred to as gel permeation chromatography, are widely employed. Thus, for example, the review article by G. Subramanian, Process scale liquid chromatography (VCH Weinheim 1995), describes preparative applications of this separation method. However, all these applications are based on batch methods, which are distinguished by a poor space-time yield. The cause is firstly the long retention times of the components, which necessitate a long cycle time (time between two injections). Since most of the materials employed in SEC have only low pressure stability, it is not possible to increase the flow rates in order to achieve faster separation. In addition, most carrier materials have poor roadability ( less than 5% of the gel volume). Some of these problems can be improved by the use of continuous methods. However, it has hitherto been impossible to apply these SEC separation methods to continuous procedures; the parameters for a continuous separation method of this type would have to be determined by suitable model calculations. In addition, the process parameters obtained must allow stable continuous operation.
Continuous chromatographic methods, such as, for example, simulated moving bed (SMB) chromatography, are traditionally employed on a large scale in the petrochemical and sugar industries. In the meantime, however, these methods have also been used in the fine chemical and pharmaceutical industries, principally for the separation of isomers and enantiomers, i.e. for separation problems of classical two-component mixtures. Initial attempts to isolate components from multicomponent mixtures have also been described.
In order to obtain suitable process parameters for SMB chromatography, a number of simulation models have been developed, of which the rigorous SMB process model has the most far-reaching approach. Simulation approaches have been disclosed, for example, by Nicoud et al. (Nancy, 1993) and in WO 97134 918. It is common to these methods that adsorption isotherms are determined for the analytes; these measurement results then form the basis of the model calculations. In recent further developments, model separations of binary mixtures have been demonstrated on the basis of the true counter current model: G. Storti et al. (1993) AlChE Journal 39, pages 471-472 and by E. Francotte et al. (1998) J. Chromatogr. A 796, pages 239-248. In a further development, the parameters have been optimised starting from a first parameter set using detailed process simulation: J. Strube, U. Altenhxc3x6ner, M. Meurer and H. Schmidt-Traub (1997) Chem.-Ing. Tech. 69, pages 328-331, and dissertation by J. Strube (University of Dortmund, 1996). However, all these models are based on the determination of adsorption isotherms.
While the chromatographic separation methods used hitherto for SMB methods, for example enantiomer separation or ion exchange chromatography, are based on adsorptive processes, SEC is based on a completely different principle: for analytes of different molecular size, different volumes are available owing to the pore size distribution in the sorbent; larger molecules thus elute earlier than small ones. SEC is thus based on a diffusive mechanism with inclusion of a size exclusion mechanism, with no adsorption of the analytes by the sorbent. There are consequently no phase equilibriums as in adsorption chromatography. The modelling methods which supply the parameters for SMB chromatography that were known hitherto thus cannot be used for SEC. In particular, the problem arises of stabilising the raffinate front of the component retained the shortest in the region of section IV, i.e. between the raffinate and eluent lines. This problem is increased further since in many SEC separation methods, part of the analytes is eluted in the void volume. Such components are transported through zone IV and contaminate the extract component. It has hitherto been impossible to apply the process parameters from batch computer models to continuous separation methods (for example SMB separation methods) for SEC separation methods, and thus specifically to obtain separation parameters for continuous separation methods which allow stable operation.
The object of the invention is thus to develop models and methods which allow separation parameters for continuous separation methods which enable stable operation to be obtained specifically for SEC separation methods.
The invention relates to continuous separation methods, particularly using SMB methods, in which the analytes are separated by size exclusion chromatography (gel permeation chromatography).